Introduction

The Celigo imaging cytometer has been developed to fully automate imaging and analysis of tumorspheres. This automated morphometric analysis tool significantly reduces the time and effort needed to quantify key aspects of 3D spheres including size, growth, growth tracking over time, and response to chemotherapeutics.

Identification and Counting of Embryoid Bodies from Spinner Flask

Experiment 1 procedure:

One mL of cultured embryoid bodies was removed a 250 mL spinner flask and plated onto a 24 well plate

The embryoid bodies were analyzed for number and average diameter

Whole-well image of embryoid bodies from a 24-well plate

Bright field image of embryoid bodies

Bright field image of counted embryoid bodies

Number of EBs

Avg. Diameter

SD Diameter

Min Diameter

Max Diameter

Celigo

643

187.1 microns

63.9 microns

82.5 microns

514.6 microns

Manual Count

81

200 microns

61.2 microns

99.6 microns

434.4 microns

The area/size of the embryoid bodies was plotted in a histogram

A gate was set to show the software identification of embryoid bodies sizes.

In this example, the red EBs have an average area of 11,577 square microns compared to the green embryoid bodies that have an average area of 38,196 square microns.

Also, this distribution shows that most embryoid bodies fall within 10 – 50k square microns with very few very large embryoid bodies that are 100k or more.

Celigo software can also plot the equivalent diameter of embryoid bodies

In this example, the size gate was set at 120 microns. Therefore, all EBs circled in red have a diameter of less than 120 microns (which is 181 EBs)

All EBs circled in blue are larger than 120 microns and are 462 in number